BIOCHEMISTRY OF FIBER TYPES OF SKELETAL MUSCLE
Fuchs, F. (1971), Biochim. Biophys. Acta 245,221.
Goodman, M., and Toniolo, C. (1968), Biopolymers 6, 1673.
Greaser, M. L.,andGergely, J. (1971), J. Biol. Chem. 246,4226.
Han, M. H., and Benson, E. S. (1970), Biochem. Biophys. Res.
Commun. 38,378.
Hartshorne, D. J., and Mueller, H. (1968), Biochem. Biophys.
Res. Commun. 31,647.
Hartshorne, D. J., and Pyun, H. Y. (1971), Biochim. Biophys.
Acta 229,698.
Metabolic Profiles of Three Fiber Types of
Skeletal Muscle in Guinea Pigs and Rabbitst
James B. Peter,* R. James Barnard, V. Reggie Edgerton, Cynthia A. Gillespie, and
Kerstin E. Stempel
ABSTRACT: Certain guinea pig and rabbit skeletal muscles,
which are composed solely or predominantly of a single type
of fiber as defined histochemically, were analyzed for various
enzymatic and substrate characteristics. The results show that
fiber types in the adult guinea pig and rabbit limb muscles
can be conveniently classified into three categories on the
basis of three criteria, (1) the contraction time of the fiber
relative to others within the same animal, (2) glycolytic capac-
ity, and (3) oxidative capacity. The rabbit semimembranosus
accessorius and the white portion of the guinea pig vastus
lateralis are fast twitch and have a very high anaerobic capac-
ity, as indicated by glycogen concentration and phosphoryl-
ase, lactate dehydrogenase and mitochondrial a-glycero-
phosphate dehydrogenase activities. Cytochrome concentra-
tion and succinate dehydrogenase activity are low, indicating
a low aerobic capacity. Consequently these fibers, formerly
termed fast-twitch white, are more explicitly called fast-
twitch-glycolytic fibers. The red portion of the guinea pig
vast us lateralis has a high glycogen concentration, moderate
lactate dehydrogenase, and high phosphorylase and a-glyc-
erophosphate dehydrogenase activities. The red vastus lat-
eralis has the highest cytochrome concentration and succinate
dehydrogenase activity, indicating a high aerobic capacity in
I n 1873 Ranvier reported that muscles which were slow
contracting appeared red whereas fast contracting muscles
appeared white. Exceptions to this general relationship be-
tween “redness” of a muscle and speed of contraction were
later reported by Denny-Brown (1929). More recent investi-
gations using a variety of histochemical techniques have dem-
onstrated that most skeletal muscles are composed of differ-
ent types of fibers and that muscles cannot be simply categor-
~~~~ ~~~~ ~~~ ~~~
t From the Neuromuscular Diseases Research Group and the
Departments of Medicine and Physical Education, University of
California, Los Angeles, Los Angeles, California 90024. Received
December 14, I971. Supported by National Institutes of Health Grants
H D 02584, NS 07587, and NS 08590. Dr. R . James Barnard is a post-
doctoral fellow of the Muscular Dystrophy Associations of America.
Murray, A. C., and Kay, C. M. (1971), Biochem. Biophys.
Res. Commun. 44,237.
Richards, E. G., Teller, D. C . , and Schachman, H. K . (1968),
Biochemistry 7,1054.
Schaub, M. C., Perry, S. V., and Hacker, W. (1972), Biochem.
J. 126,237.
Shapiro, A. L., Vinuela, E., and Maizel, J. V. (1967), Biochem.
Biophys. Res. Commun. 28,815.
Yphantis, D. A. (1964), Biochemistry 3,297.
addition to moderate to high glycolytic capacity. Conse-
quently these fibers, previously labeled fast-twitch red, can more
explicitly be called fast-twitch-oxidative-glycolytic fibers.
Although this fiber type is found in the rabbit, no muscle
consisting predominantly of fibers that are fast-twitch-oxida-
tive-glycolytic was found. The soleus muscle is slow twitch
and chemically is characterized by low glycogen concentra-
tion and low phosphorylase, lactate dehydrogenase, and
mitochondrial a-glycerophosphate dehydrogenase activities
together with a moderate cytochrome concentration and suc-
cinate dehydrogenase activity. These features indicate a mod-
erate to high aerobic and a relatively low glycolytic capacity.
Formerly labeled the slow-twitch intermediate fiber they are
more appropriately called slow-twitch-oxidative fibers. These
data provide quantitative information on the metabolic
characteristics of the three distinct muscle fiber types pre-
viously described by histochemical techniques. Furthermore,
since muscles composed of fast-twitch-oxidative-glycolytic
or slow-twitch-oxidative fibers are red in appearance the
classification of skeletal muscle solely as “red” and “white”
is imprecise and incomplete. Failure to recognize this can
complicate the interpretation of studies with skeletal muscle.
ized as “red” or “white” (Barnard et al., 1971 ; Edgerton and
Simpson, 1969; Guth and Samaha, 1969; cf Peter, 1972).
Histochemical, biochemical, and physiological studies of
hind-limb skeletal muscle of guinea pigs have demonstrated
the presence of three fiber types, each of which possesses a
distinctive combination of histochemical and mechanical
characteristics (Barnard et al., 1971). These three fiber types
were classified as fast-twitch red, fast-twitch white, and slow-
twitch intermediate based on (1) their twitch characteristics
which correlate with the specific activity of myosin ATPase
and (2) histochemical assessment of their oxidative and gly-
colytic capacities (Edgerton and Simpson, 1969; Gillespie
e f al., 1970; Barnard et al., 1971;Peter, 1972).
The purpose of this paper is to provide quantitative data on
the enzyme activities and glycogen concentration in muscles
BIOCHEMISTRY, VOL. 11, NO. 14, 1 9 7 2 2627

composed predominately or exclusively of one of these types
of fibers. Based on the data obtained and on the histochemical
and contractile properties described earlier (Edgerton and
Simpson, 1969; Barnard et al., 1971; CJ Peter, 1972, for re-
view) a more explicit nomenclature is proposed in which the
terms fast-twitch white, fast-twitch red, and slow-twitch
intermediate are replaced by fast-twitch-glycolytic, fast-
twitch-oxidative-glycolytic, and slow-twitch-oxidative, re-
spectively.
Methods
Muscle Preparation. The muscles used in this study were
obtained from adult guinea pigs and rabbits. The soleus
( 1 0 0 z slow-twitch-oxidative fibers), the red portion of the
vastus lateralis ( 7 8 z fast-twitch-oxidative-glycolytic fibers),
and the white portion of the vastus lateralis (71 % fast-twitch-
glycolytic fibers) were taken from the guinea pig. The soleus
z
(96 slow-twitch-oxidative fibers) and the semimembranosus
accessorius ( 8 6 z fast-twitch-glycolytic fibers) were taken
from the rabbit. The guinea pigs were sacrificed by a sharp
blow to the head and the rabbits by intravenous injection of
air. The muscles were quickly removed, trimmed, and weighed.
Muscles used for cytochrome, succinate dehydrogenase, and
mitochondrial a-glycerophosphate dehydrogenase assays
were immediately frozen in liquid nitrogen and stored at -70'
for 3-6 days. Preliminary experiments showed no loss in en-
zyme activity with short periods of freezing. Myoglobin
determinations were also done on frozen muscle.
For the determination of hexokinase and lactate dehydro-
genase fresh muscle was homogenized in cold (1-4") buffer
medium (pH 7.4) containing 50 mM Tris, 1 mM EDTA, 15
mM K2S04,and 6 mM MgC12. Fresh muscles used for phos-
phorylase determinations were homogenized in 100 mM Tris
(pH 7.4) and muscles used for a-1,4-glucosidase determina-
tions were homogenized in distilled water. For the deter-
minations of cytochromes, succinate dehydrogenase, and
mitochondrial a-glycerophosphate dehydrogenase the frozen
muscles were thawed and homogenized in a buffer medium
(pH 7.4) containing 50 m~ Tes, 100 mM KC1, 5 mM MgS04,
1 mbi EDTA, and 50 mM KPi.
Immediately after sacrifice the trimmed muscles to be as-
sayed for glycogen were placed in hot KOH (373 for glyco-
gen extraction and subsequent acid hydrolysis (Gillespie
et al., 1970). Glucose was then measured according to the
method of Nelson (1944).
For myoglobin determinations, the muscles were thawed,
homogenized with sand in a mortor and dialyzed overnight
against 0.1 M Tris (pH 8.2). Supernatants were then chromato-
graphed on a Cellex D (DEAE) column and myoglobin was
determined according to Brown (1961) with the following
exceptions. The precipitation of myoglobin with ammonium
sulfate was eliminated and the supernatant from the first
centrifugation was dialyzed with Tris buffer for 24 hr and
then separated by column chromatography.
Enzyme Assays. All enzyme activities were determined on a
Gilford 2000 recording spectrophotometer at 37" and are
expressed as initial maximum rates.
1 Anatomy and abbreviations used are: exact location of the semi-
membranosus accessorius is described by Kerr (1955). The small muscle
located in the middle of the semimembranosus accessorius is the semi-
membranosus proprius and is composed predominately of slow-twitch-
oxidative fibers: Tes, N-tris(hydroxymethyl)methyl-2-aminoethane-
sulfonic acid.
2628 BIOCHEMISTRY, VOL. 11, NO. 14, 1 9 7 2
PETER e t al.
Hexokinase (EC 2.7.1.1) activity was measured by a modi-
fication of the method of Sharma et al. (1963). This method
is based on the rate of NADPH formation in a medium con-
taining 50 mht Tris, 2.25 mM K2S04,8 mhi MgCl?, 3 mhi ATP.
0.2 mM EDTA, 0.75 mM NADP, 2.5 mM glucose, and e x c r ~ ,
glucose-6-phosphate dehydrogenase and 6-phosphogluconate
dehydrogenase. Measurements were done on both the gauze-
filtered homogenate and 105,000gsupernatant.
Phosphorylase (EC 2.4.1.1) activity was determined in a
medium (pH 7.4) containing 75 mM Tris, 5 nib1 MgCI?, 0.5
mM NADP, 10 m~ KPi, 0.6 mhi AMP, 1 . 3 z glycogen, and
excess glucose-6-phosphate dehydrogenase and phosphoglu-
comutase. Measurements were made on the 270g supernatant
which contains all of the phosphorylase activity. This assay
medium measures total phosphorylase activity.
Lactate dehydrogenase (EC 1.1.1.27) activity was measured
as the initial rate of oxidation of NADH at pH 7.4 in a me-
dium containing 50 mM KP;, 0.45 mhi NADH, and 0.35 m u
sodium pyruvate. Determinations were done on the 105,OOOg
supernatant which contained all of the lactate dehydrogenase
activity.
Succinate dehydrogenase (EC 1.3.99.1) activity was mea-
sured according to a modification of the ferricyanide method
of Bonner (1955). The gauze-filtered whole homogenate was
first preincubated at 37' for 5 min to deplete the endogenous
substrate. The reaction mixture (pH 7.4) contained 0.1 11
KPi, 10 miv KCN, 1 mM K3Fe(CN)8, and 20 mhi sodium
succinate. Rotenone (20 p ~ was ) added to block the oxidation
of NADH.
a-Glycerophosphate dehydrogenase (EC 1.1.2.1) activity
was measured by the ferricyanide method described by Bass
et al. (1969). The gauze-filtered homogenate was preincubated
for 5 min at 37". The reaction mixture contained 0.1 M KP;
(pH 7.4), 5 mM EDTA, 10 mM KCN, 1 mM K3Fe(CN)c,0.4 g
of bovine albumin/100 ml, 25 mbi a-glycerophosphate, and
20 p~ rotenone.
a-I,4-Glucosiduse (EC 3.2.1.20) activity was determined
by the method described by Hudgson et ul. (1968). The homog-
enate was incubated at 37" with shaking in a medium con-
sisting of 5 mhi maltose in 0.1 hi acetate (pH 4.0). Aliquots
were removed from the reaction mixture at 30 min intervals
and immediately placed in 1 2 % HC104. The solution was
then neutralized with KsC03 and centrifuged at 10,OOOg for
10 min. The amount of glucose formed was determined by the
maximum change in optical density when an aliquot was
added to a medium containing 100 mhi Tris (pH 7.4), 5 mhi
MgC12, 0.5 mM NADP, 1 mM ATP, and excess hexokinase
and glucose-6-phosphatedehydrogenase.
Cytochromes a and c concentrations were measured by the
difference spectra method of Schollmeyer and Klingenberg
(1962). Complete oxidation of the cytochromes was produced
in a test tube containing 2 phi carbonyl cyanide nz-chloro-
phenylhydrazone, and 1.5 ml of homogenate (1 :7. w;v).
To a second test tube containing 2 p~ carbonyl cyanide in-
chlorophenylhydrazone and 8 mM sodium succinate, 1.5 ml
of homogenate was added and shaken for 1 min; freshly
prepared potassium cyanide (2.5 mM) was then added and the
solution incubated at 37" for 5 min. Both tubes were gassed
for 30 sec with 100% Onand the difference spectra then re-
corded on a Cary 14 spectrophotometer. The difference in
optical density between the completely oxidized and coni-
pletely reduced forms was determined at the following wave
lengths: cytochrome c + cl, 550-541 mp, and cytochrome
a, 605-630 mp. Extinction coefficients of 14 and 18 were used
for cytochromes a and c , respectively.
BIOCHEMISTRY OF FIBER TYPES OF SKELETAL MUSCLE

TABLE I : Guinea Pig Skeletal Muscle Characteristics.

Muscle Red Vastus White Vastus Soleus

Gross appearance Red White Red
Histochemical fiber type (%)"
Fast-twitch-oxidative-glycolytic 78 f 2 29 f 2 0
Fast-twitch-glycolytic 18 f 2 71 f 0 . 6 0
Slow-twitch-oxidative 4 f 0.5 0.0 100
Time-to-peak tension (msec)" 19.0 f 0 . 4 20.1 f 0 . 4 82.3 * 1.3
ATPase (pmoles/min per mg of natural
actomyosin), pH 9 . 4
Actomyosin 0.16 f 0.02 0.27 f 0.01 0.04 f 0.01
Myosin 0.34 f 0.02 (6) 0.32 i 0.01 (6) 0.14 f 0.01 (6)
Glycogen' (mg/g wet weight) 9 . 7 f 0 . 8 (11) 7 . 4 f 0 . 6 (11) 3 . 3 i 0 . 4 (5)
Phosphorylase (pmoleslmin per g)
270g supernatant 6.67 f 0.66(8) 7.13 * 0.72 (7) 1.54 f 0 . 0 9 (6)
Lactate dehydrogenase (pmoles/min/g),
105,OOOg supernatant 218 i 19(9) 449 f 41 (7) 105 f7 (6)
a-Glycerophosphate dehydrogenase
(pmoles/min per g)
Whole homogenate 1.67 f 0.16(6) 1.62 f O.lO(6) 0.61 i 0.05 (6)
Hexokinase (nmoles/min per g)
Whole homogenate 620 f 54 (6) 297 f 57 ( 5 ) 978 f 59 (4)
105,OOOg supernatant 177 f 17 71 f 11 358 f 30
a-l,4-Glucosidase (nmoles/min per g)
Whole homogenate 22.1 f 1 . 9 (4) 9.9 f 0.7 (4) 25.5 f 1.0(5)
Succinate dehydrogenase (pmoles/min
Per 8)
Whole homogenate 2.49 f 0.29 (6) 0.72 f 0.05 (5) 1.95 f 0.17 (4)
Cytochrome a (nmoles/g)
Whole homogenate 12.8 f 0 . 7 (5) 2 . 2 i 0 . 3 (7) 4 . 8 f 0 . 5 (4)
Cytochrome c (nmolesig)
Whole homogenate 18.0 f 0 . 9 (5) 1 . 9 i 0 . 3 (7) 6 . 5 f 0 . 3 (4)
Myoglobin (mg/g) 1.44 f 0.20 (14) 0.31 f 0.07 (5) 1.39 i 0 . 1 5 (8)
a Data cited from previous publications (Barnard et af.,1971 ; Gillespie et af.,1970). Values are means f std dev. The number
of muscles analyzed is given in parentheses.

Results vastus of the guinea pig and the semimembranosus accesso-

Table I summarizes the observations made on the three
different guinea pig muscles. The red vastus, white vastus,
and soleus are composed predominately of fast-twitch-oxida-
tive-glycolytic, fast-twitch-glycolytic, and slow-twitch-oxida-
tive fibers, respectively, as determined by the histochemical
activity of myofibrillar ATPase, mitochondrial a-glycero-
phosphate dehydrogenase, NADH-diaphorase, and malate
dehydrogenase (Figure le-h). The specific activity of myosin
ATPase measured in homogenates of these muscles is con-
sistent with physiological and histochemical findings (Table
I ; Figure 1; Barnard et al., 1971) in that muscles with fast
contraction times have a higher specific activity of myosin
ATPase and stain darker at pH 9.4 for myofibrillar ATPase
than do the slow-twitch muscles (Tables I and 11).
The serial sections of rabbit (Figure la-d) and guinea pig
(Figure le-h) gastrocnemius show that fibers staining darkest
with myofibrillar ATPase also stain darkest with mitochon-
drial a-glycerophosphate dehydrogenase and phosphorylase
(not shown) and lightest with NADH-diaphorase and malate
dehydrogenase (Figure 1). These fibers are histochemically
similar to the fast-twitch-glycolytic fibers found in the white
rius of the rabbit.
By contrast slow-twitch-oxidative fibers stain negligibly
for myofibrillar ATPase at pH 9.4 and for mitochondrial
a-glycerophosphate dehydrogenase whereas the histochemical
activity of NADH-diaphorase and malate dehydrogenase is
intermediate between that of the fast-twitch-oxidative-gly-
colytic and slow-twitch-oxidative fibers (Figure 1). In addi-
tion to these differences the distribution of reaction product
with NADH-diaphorase, malate dehydrogenase, and succi-
nate dehydrogenase in slow-twitch-oxidative fibers is uniform
throughout the fiber cross-section and its size is fine in con-
trast to the subsarcolemmal preponderance of coarse reac-
tion product in fast-twitch-oxidative-glycolytic and fast-
twitch-glycolytic fibers (Figure 1 ; Table 111). In the sections
shown, this difference in pattern is most obvious with malate
dehydrogenase (Figure ld,h).
As illustrated in Figure la-d the third type of fiber termed
fast-twitch-oxidative-glycolytic stains for myofibrillar ATPase
and mitochondrial a-glycerophosphate dehydrogenase with
an intensity between that of fast-twitch glycolytic (dark) and
slow-twitch-oxidative fibers (very light) but stains most in-
tensely for NADH-diaphorase and malate dehydrogenase.

B I O C H E M I S T R Y , VOL. 1 I, NO. 14, 1 9 7 2 2629

 P E T E R f?t U!.

TABLE 11: Rabbit Skeletal Muscle Characteristics.

._. _ _ ~ _ _ _ _ _ _ ._

Muscle
Semimembranosus
Accessorius Soleus
Gross appearance White Red
Histochemical fiber type (%)"
Fast-twitch-oxidative-gly colytic 12 4
Fast-twitch-glycolytic 86 0
Slow-twitch-oxidative 2 96
Time-to-peak tension (msec) 30 85
Phosphorylase (pmoles/min per g)
270g supernatant 8 25 + 1 72 (4) 2 06 * 0 31 (4)
Lactate dehydrogenase (pmoles/min per g)
105, OOOg supernatant 1485 i 154 (5) 328 i 88 (5)
a-Glycerophosphate dehydrogenase (pmoles/min per g wet wt)
Whole homogenate 1 46 & 0 31 (4) 0 28 =k 0 14 (4)
Hexokinase (nmoles/min per g)
Whole homogenate 183 + 53 (7) 993 i 115 (7)
Succinate dehydrogenase (pmoles/min per g wet wt)
Whole homogenate 0 64 =k 0 14 (4) 1 81 k 0 16 (4)
Cytochrome LI (nmoles/g wet wt) < 1 O(4) 7 0 i 0 6(5)
Cytochrome c (nmoles/g wet wt)
Whole homogenate <1 0 (4) 7 3 I 0 6(4)
~~ ~

a These data are estimates taken from small samples of each of the three muscles. Values are means =tstd dev. The number of
muscles analyzed is given in parentheses.

TABLE I11

Type of Fiber
~ ~

Fast-Twitch-Oxidative- Fast-Twitch- Slow-Twitch-

Glycolytic Glycolytic Oxidative
Staining intensity
NADH-Diaphorase High Low Intermediate
Malate dehydrogenase High Low Intermediate
Phosphorylase High High Low
PAS High High Low
Mitochondrial a-glycerophosphate dehydrogenase High High Low
Myofibrillar ATPase, pH 9.4 Intermediate-High High Low
Staining with NADH-diaphorase
Cytological localization Many Few More uniform
subsarcolemmal su bsarcolemmal throughout fiber
aggregates aggregates
Granular size of stained particles Large Large Small
Previous terminologies
Reference
Dubowitz and Pearse (1960), Engel (1962) I1 TI I
Romanul(l964) 11 I I11
Yellin and Guth (1970) ffP ff @
Ashmore and Doerr (1971) a-red a-white @-red
Brooke and Kaiser (1970) IIA IIB I
Stein and Padykula (1962) C A B
Padykula and Gauthier (1966) Red White Intermediate
Burke et al. (1971) FR FF S

Histochemical activities of phosphorylase, lactate dehydro- oxidative-glycolytic. These fibers in the guinea pig gastroc-
genase, and succinate dehydrogenase also show that this nemius are presumed to be fast because of their great histo-
fiber has more glycolytic and oxidative activity than the slow- chemical similarity to the predominant fiber of the red vastus,
twitch-oxidative fiber. Hence, it is described as fast-twitch- a muscle of the guinea pig known to be fast-twitch and com-

2630 BIOCHEMISTRY, V O L . 11, NO. 14. 1 9 7 2

B I O C H E M I S T R Y Of F I B E R T Y P E S O F S K E L E T A L M U S C L E

posed predominately of such fibers. Table 111 correlates the

histochemical characteristics of these fibers with an outline
of previous terminologies employed to describe various types
of fibers.
The histochemical activity of myofibrillar ATPase at pH
9.4 in the three fibers correlates with the specific activity of
natural actomyosin ATPase (Figure 1 and Table I); the fast-
twitch-oxidative-glycolytic fibers show more activity than the
slow-twitch-oxidative but less than fast-twitch-glycolytic
fibers.
In the guinea pig the red vastus and white vastus both have
high glycogen concentrations and high phosphorylase, lactate
dehydrogenase, and mitochondrial a-glycerophosphate de-
hydrogenase activities relative to the soleus (Table I). These
muscles are composed predominately of fast-twitch-oxidative-
glycolytic, fast-twitch-glycolytic, and slow-twitch-oxidative
fibers, respectively (Table 111). Hexokinase activity which is
highest in the soleus does not parallel either the glycolytic
activities or Cytochrome concentrations. Mitochondrial a-
glycerophosphate dehydrogenase correlates with the gly-
colytic activities rather than with succinate dehydrogenase
or cytochrome concentration (Table I). Like hexokinase the
activity of a-1,4-glycosidase is high in both the soleus and
red vastus relative to the white vastus. Succinate dehydro-
genase activity and cytochrome concentrations are highest
in the red vastus, intermediate in the soleus, and lowest in
the white vastus. Myoglobin concentration is high in both the
red vastus and soleus and is very low in the white vastus.
Table I1 summarizes the observations made on the two
different rabbit muscles composed predominately of fast-
twitch-glycolytic and slow-twitch-oxidative fibers. The popu-
lation distribution of individual fibers in these muscles is an
approximation made from histochemical examination of
three each of the different muscles. Although the absolute
level of enzyme activities between the two species show some
FIGURE I: Rabbit la-d and guinca pig le-h gastrocncmius muscles
quantitative differences, the enzyme profiles of fast-twitch- consisting of the three basic fiber types are illustrated. Myofibrillar
glycolytic and slow-twitch-oxidative fibers in the rabbit are ATPase after an alkaline buffer preincubation and alkaline incuba-
similar to those observed in the guinea pig, as are the histo- tion at pH 9.4 (a$), mitochondrial a-glycerophosphate dehydro-
enzymatic activities of the three fiber types (Figure 1). No genase (&f), reduced nicotinamide adenine dinucleotide diaphorase
hind limb muscle composed predominately of fast-twitch- (c,g),and malate dehydrogenase (d,h)are shown. Fibers that are the
darkest staining with ATPase (fast twitch) have high a-glycerophos-
oxidative-glycolytic fibers was found in the rabbit. phate dehydrogenase and low NADH-diaphorase and malate
dehydrogenase activity and are called fast-twitch-glycolytic fibers.
Other fibers that stain darkly with ATPase (but slightly less than in
Discussion fast-twitch-glycolytic fibers) stain moderately with n-glycerophos-
phate dehydrogenase and heavy with NADH-diaphoraseand malate
The biochemistry of red and white muscle has been of in- dehydrogenaseare called si st-twitch-oxidative-glycolytic. The fibers
terest to scientists since the early 1900's. Early studies of the which show the lightest n.yofibrillar ATPase activity also have the
biochemical, structural and functional characteristics of red lowest mitochondrial n-glycerophosphate dehydrogenase activity
and white muscle were reviewed by Needham (1926). Pette and moderate NADH-diaphorase and malate dehydrogenase activ-
ity and are called slow-twitch-oxidative. Morphologically fast-
and his associates (Pette and Bucher, 1963) and Beatty, Bocek, twitch-oxidative-glycolytic and slow-twitch-oxidative fibers differ
and their associates (Beatty and Bocek, 1970) have added ex- in that the granularity of the staining is more coarse and localized
tensive biochemical data on red and white muscle in different more peripherally in the former. Although not illustrated here histo-
species. chemical phosphorylase activity as well as glycogen concentration by
Unfortunately the design of most of these biochemical PAS stain are higher in the fast-twitch-oxidative-glycolytic and fast-
twitch-glycolytic than in slow-twitch-oxidative fibers of the guinea
investigations indicates a reliance on muscle color and a lack pig (Gillespie et al., 1970). The bar in Figure la represents 50 N.
of application of information obtained from numerous histo-
chemical studies describing the existence of at least three
distinct types of skeletal muscle fibers (cf:Table 111). Although
Beatty and Bocek (1970) mention an intermediate fiber, they profile including oxidative and glycolytic enzyme activities.
were unable to assess its biochemical properties because The relative speeds (times-to-peak tension) of hind limb
muscles composed predominately of one fiber type were not muscles composed predominately or exclusively of one or
studied. another histochemical type of fiber determine their classifica-
In the past we have classified the three fiber types found in tion as fast-twitch or slow-twitch (Edgerton and Simpson,
mammalian hind limb muscles as fast-twitch red, fast-twitch 1969;Barnardetal., 1971;Peter, 1972).
white, and slow-twitch intermediate on the basis of their con- Based on the quantitative data presented herein we now
traction time and according to their histochemical enzyme suggest that shorthand, nondescriptive nomenclatures such as

BIOCHEMISTRY, VOL. 11, NO. 14, 1 9 7 2 2631


are listed in Table I11 as well as our own imprecise designation
of fibers as fast-twitch white, fast-twitch red, and slow-twitch
intermediate be replaced by the straightforward, descriptive
terms fast-twitch-glycolytic, fast-twitch-oxidative-glycolytic,
and slow-twitch-oxidative. The latter classification is purpose-
fully open-ended so that additional terms characterizing simi-
larities or differences between the fibers can be appended.
In addition, the precision of the terms themselves can be
improved by quantifying the terms used, e.g., by appending
to the speed term the measured time-to-peak tension of such a
fiber or of a muscle composed of such fibers.
This system of nomenclature readily accommodates other
permutations and combinations of muscle speed and met-
abolic pattern which are possible such as fast-twitch-oxidative,
slow-twitch-glycolytic, and slow-twitch-oxidative-glycolytic,
some of which are expected in various species. Indeed fast-
twitch-oxidative fibers are known to exist in pigeon extra-
ocular muscles (Maier et al., 1972) and probably in the rabbit
cricothyroid (Hall-Craggs, 1968). In addition the classifica-
tion should prove useful for describing tonic, nonpropagating
fibers as well as fibers of muscle spindles.
We would emphasize, however, that at this point there
can be no definite answer to the fundamental question of
whether the fast-twitch-glycolytic fibers and fast-twitch-
oxidative-glycolytic fibers described herein reflect quantum
differences in metabolic properties of two distinct popula-
tions of fibers or whether they are simply convenient examples
for description of what is really a continuum of metabolic
properties of fast-twitch fibers in certain hind-limb skeletal
muscles of the species studied. An answer to this question
should be possible with analysis of the biochemical, histo-
chemical and quantitative electron microscopic features of
many individual fast-twitch fibers. In any case investigations
of skeletal muscle must take into account the differences in
fast-twitch muscle fibers.
The present study defines certain quantitative biochemical
differences in muscles known to be composed predominately
or exclusively of a single fiber type. The enzyme activities
given in Tables 1 and I1 represent maximum activities mea-
sured in aitro under the conditions described. Although the
activity of these enzymes may be different in ciGo due to com-
partmentalization, substrate-product inhibition, etc., the
in vitro data establish the gross metabolic capabilities of slow-
twitch-oxidative, fast-twitch-glycolytic, and fast-twitch-oxida-
tive-glycolytic muscle fibers in the guinea pig as well as siow-
twitch-oxidative and fast-twitch-glycolytic fibers in the rabbit.
These capabilities are expected to parallel the in cico capacity
for glycogenolysis, glycolysis, and oxidative metabolism, but
further studies are needed to establish the precision of this
relationship.
Fast-twitch-glycolytic fibers (guinea pig, white vastus lat-
eralis ; rabbit, semimembranosus accessorius) are predom-
inately anaerobic fibers. They have a high glycogen concen-
tration and high phosphorylase, lactate dehydrogenase and
a-glycerophosphate dehydrogenase activities. Their aerobic
capacity is very limited as manifest by low succinate dehydro-
genase activity as well as low cytochrome and myoglobin con-
centrations. The high activities of phosphorylase, lactate de-
hydrogenase, and mitochondrial a-glycerophosphate dehy-
drogenase relative to cytochrome u concentration (Tables I
and 11) also indicate that fast-twitch-glycolytic fibers rely
mainly on anaerobic metabolism for the production of ATP.
Fast-twitch-oxidative-glycolytic fibers (guinea pig, red
vastus lateralis) appear to have the highest capacity for aero-
bic metabolism because succinate dehydrogenase activity as
2632 BIOCHEMISTRY, VOL. 11, NO. 14, 1 9 7 2
PETER e t nl.
well as cytochrome and myoglobin concentrations are great-
est in these fibers. Fast-twitch-oxidative-glycolytic fibers are
also characterized by a moderate to high glycolytic capacity.
They have the highest glycogen concentration and moderate
lactate dehydrogenase activity. In these fibers of the guinea
pig the phosphorylase and mitochondrial a-glycerophosphate
dehydrogenase activities are very high. The high a-glycero-
phosphate dehydrogenase activity suggests an important role
for the a-glycerophosphate shuttle system in the regeneration
of NAD for glycolysis in both fast-twitch-glycolytic and fast-
twitch-oxidative-glycolytic fibers. The latter fibers in the
guinea pig also have a high capillary to fiber ratio in compar-
ison to fast-twitch-glycolytic fibers, suggesting a blood flow
adequate for aerobic metabolism (Mai et a[., 1970). Other
investigators have found the myoglobin concentration in cat
muscle correlates directly with blood flow and inversely with
speed of contraction (Reis and Wooten, 1970). Our work
supports the direct relationship between capillary density
and myoglobin concentration but study of fast-twitch-oxida-
tive-glycolytic fibers clearly shows that the relationship be-
tween capillary or myoglobin concentration and speed of con-
traction is not necessarily inverse (Mai et id,,1970; Table I).
Other studies from this laboratory show quantitative differ-
ences in the ATPase activity and other characteristics of
natural actomyosin isolated from fast-twitch-oxidative-gly-
colytic fibers compared to that from fast-twitch-glycolytic
fibers (Table I ; Furukawa and Peter, 1971). In particular the
specific activity of the Mg2+-activated actomyosin ATPase
at pH 9.4 is highest in fast-twitch-glycolytic fibers, intermedi-
ate in fast-twitch-oxidative-glycolytic fibers, and lowest in
slow-twitch-oxidative fibers (Table I). These data (Table I)
correspond to the intensity of staining of the same fibers for
myofibrillar ATPase (Figure 1 and Table 111) and suggest
that a component of the thin filament contributes to the histo-
chemical differentiation of the fast-twitch fibers. On the other
hand, the characteristics of calcium transport by fragmented
sarcoplasmic reticulum isolated from fast-twitch-oxidative-
glycolytic and fast-twitch-glycolytic fibers (the red vastus and
white vastus, respectively) are very similar (Fiehn and Peter,
1971).
The high capacities for glycogenolysis, glycolysis, and oxi-
dative phosphorylation together with its speed of contraction
give the fast-twitch-oxidative-glycolytic fiber a distinctive
metabolic profile. The magnitude and scope of these metabolic
activities appear to gear these fibers for high intensity, pro-
longed work, a proposed function which is supported by
their selective recruitment during exercise (Edgerton er al.,
1970a,b).
The enzyme profile of fast-twitch-oxidative-glycolytic
fibers could not be quantitated in homogenates of rabbit
muscle, because muscle composed predominately of such
fibers was not found. However, fast-twitch-oxidative-gly-
colytic fibers, identified histochemically in heterogeneous
hind limb muscles of the rabbit, have a n enzyme profile deter-
mined histochemically which is in complete agreement with
results found in homogenates of fast-twitch-oxidative-gly-
colytic muscles in the guinea pig.
Slow-twitch-oxidative fibers (guinea pig and rabbit soleus)
appear to rely predominately on aerobic metabolism. These
fibers have a low glycogen concentration and low phospho-
rylase, lactate dehydrogenase, and mitochondrial a-glycero-
phosphate dehydrogenase activities. Their cytochrome con-
centration and succinate dehydrogenase enzyme activity are
intermediate between those observed in the fast-twitch-oxi-
dative-glycolytic and fast-twitch-glycolytic fibers.
BIOCHEMISTRY OF FIBER TYPES OF SKELETAL MUSCLE
In addition to these metabolic differences among the three
fiber types other work showed different activities of acid
hydrolases (Peter et al., 1972) and different isoenzyme patterns
of lactate dehydrogenase in the three fiber types with a full
complement of all five isoenzymes in muscles composed pre-
dominately of fast-twitch-oxidative-glycolytic fibers (Peter
et al., 1971 ; Sawaki and Peter, 1972).
The biochemical and histochemical data reported in this
paper, in addition to histochemical data already published,
adequately demonstrate the existence of three muscle fiber
types in hind-limb muscles of the giiinea pig and rabbit. Histo-
chemical data support the contention that .these three met-
abolically distinct fiber types are present also in hind-limb
muscles of the mouse, rat, cat, rabbit, and lesser bush baby.
It is important to recognize, however, that the magnitudes of
the aerobic systems are not static and can be changed in a
given type of fiber with physiological stimuli such as exercise
(Holloszy, 1967; Barnard et al., 1970; Peter, 1972; Barnard
and Peter, 1971). For this reason the quantitative aspects of
the energy-generating systems of one fiber compared to an-
other (e.g., the cytochrome concentration in slow-twitch-
oxidative fibers compared to that of the cytochrome-rich
population of fast-twitch-oxidative-glycolytic fibers) should
be expected to vary from species to species and perhaps even
within the same species under different conditions or in differ-
ent muscles. Such variations emphasize our contention that
hind-limb skeletal muscles are best classified as fast twitch or
slow twitch and subclassified according to as many metabolic
features as possible. The latter features are more mutable
and doubtless reflect the type, intensity, and duration of work
required of a given fiber in a given muscle.
Since most muscles are composed of a mixture of the three
fiber types, it is important to know the exact composition
of muscles used. In biochemical studies of skeletal muscles
selection of muscles composed predominately or exclusively
of one fiber type is mandatory if unnecessary obfuscation
is to be avoided.
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